Soluble contaminants may be present in a purified product sample in trace amounts that are difficult to detect by conventional means, but which may have significant physiological effects. Thus, the presence of trace solute impurities often needs to be determined during or after synthesis or purification of the product. It may be necessary to assess the quality of a product preparation, including the presence of trace solute impurities, at each step of a preparative procedure and at the end of the preparative procedure when the product is ready for use. Furthermore, federal regulations mandate purity specifications for many consumer products, particularly foods and pharmaceutical products. Generally, the quality of a product preparation is determined using a previously established criterion for identification, for example, a characteristic unit activity. If the product of interest is a protein, identification also may be by molecular weight, cryptic digest/peptide mapping, and/or immunoaffinity. The presence of soluble trace contaminants in the product preparation is often masked by the presence of the product itself, particularly if the product comprises a highly concentrated preparation.
Detection of trace solute impurities in a product preparation may be difficult, undesirably time-consuming, and even impossible without wasting large amounts of the product if the amount of product present in the sample is greatly in excess of the amount of impurity present. This can be a problem where minute amount of impurities present in a medically useful product can cause serious physical side-effects when administered to a patient. Detection of the trace contaminants should be accurate, rapid, adaptable, and repeatable.
Trace solutes have been detected using polyclonal antisera raised against the background components of a recombinant protein mix. For example, if the recombinant protein is produced in bacteria, antisera can be raised against the total proteins from an identical bacterial strain that has not been transformed with DNA encoding the recombinant protein. This antiserum will detect bacterial proteins only and not the recombinant product. However, the limit of sensitivity of an immunoassay utilizing this type of antiserum is the limit of sensitivity of the immunoassay itself (i.e., 10.sup.-12 moles). Also, the repeatability of such an assay exploiting polyclonal antisera raised to a complex mixture of antigens is extremely poor.
Chromatographic and electrophoretic techniques are well known in the art as means for separating components (solutes) present in a mixture. These techniques are particularly useful in the chemical and biotechnological arts. True chromatography describes the separation of solutes according to their different partitioning between two (or three) phases. The phases generally are solid and liquid, and solute partitioning results in their differing mobilities through a layer of solids, typically particulate, matrix in the presence of a flowing phase. Solute transfer through the layer may be along a pressure gradient, generally referred to as "liquid chromatography". In contrast, electrophoretic systems separate solutes on the basis of their electrophoretic mobility, isoelectric point, and/or differential migration through a size discriminating matrix. Solute transfer in these systems is driven by a voltage gradient from an applied electric field.
Chromatographic matrices can separate components by any of a number of criteria, including size, electrical charge, hydrophobic interaction, and/or specific affinity for the matrix or binding sites thereon. Because the components in the mixture will vary in their affinity for the matrix, their partitioning as they pass through the matrix separates the components so that they exit the matrix sequentially, separated temporally and spatially. Determination of the location of the various separated components, or of a given component of interest within the sequence, generally is achieved by collecting the fluid phase exiting the matrix (i.e., the effluent stream) as a series of fractions and sampling these fractions to identify their contents by any of a number of means known in the art.
Resolution of the various components in the mixture depends on several considerations, chief among them being the partitioning ability of the matrix and the system's theoretical plate height and plate number (see infra). In general, a large surface area-to-volume ratio is desired. Matrices for liquid chromatography systems typically are housed in cylindrical chromatography systems known as columns. In electrophoresis systems, high resolution also demands efficient removal of the heat generated by the applied electric field. Capillary electrophoresis, or other electrophoretic modules which provide a large surface area-to-volume ratio dissipate Joule heat well, allowing rapid analysis without significant loss of resolution.